The cross-linking of mast cell-bound IgE upon binding of IgE to allergens induces Type I allergic diseases such as atopic dermatitis and atopic asthma. Diseases related to allergy and atopy affect a significant percentage of the population, including up to 20% of humans, and are increasing every year. A significant proportion of type I allergic patients are mite allergic. For example, based on skin tests, at least 75% of the estimated 50 million asthmatics in the United States are mite allergic. As such, mite proteins comprise an important allergen in type I allergic disease. The house dust mites Dermatophagoides farinae and Dermatophagoides pteronyssinus are the most common mites in the United States. These mites produce several classes, or groups, of allergens, one of which is known as Group 1 proteins, which are also found in other mite species. For example, considerable cross-reactivity has been found among Blomia tropicalis, D. farinae and Lepidoglyphus destructor allergens; see, for example, Colloff, 1993, J Allergy Clin Immunol 91, 1042-1050. Additionally, Group 1 proteins have been found in D. pteronyssinus, D. farinae, Euroglyphus maynei, and L. destructor, see, for example, Thomas et al, 1998, Allergy 53, 821-832.
Mite Group 1 proteins share significant homology with a family of cysteine proteases including actinidin, papain, cathepsin H and cathepsin B. These Group 1 proteins are commonly found in the feces of mites and are thought to function as digestive enzymes in the mite intestine. Group 1 proteins from different mites are highly homologous, approximately 25-kilodalton (kD) secretory glycoproteins, that are synthesized by the cell as a pre-pro-protein that is processed to a mature form. D. farinae, D. pteronyssinus, and E. maynei Group 1 proteins, for example, share about 80% identity. In particular, Group 1 proteins from D. farinae and D. pteronyssinus, also referred to as Der f 1 and Der p 1 proteins, respectively, show extensive cross-reactivity in binding IgE and IgG. In human populations that are mite allergic, approximately 80% to 90% have IgE that is reactive to Group 1 proteins; see Thomas, 1996, Adv Exp Med Biol 409, 85-93.
Since Group 1 proteins are important in mite allergy, it is desirable to have sufficient quantities of these proteins for uses related to diagnosing and treating diseases that are related to mite allergy. To obtain the amounts of Group 1 proteins necessary for these purposes, it is desirable to use recombinant expression systems, since the amount of Group 1 proteins in mites is relatively small and the purification process to obtain native Group 1 proteins is a difficult, multi-step process. Nucleic acid molecules encoding Der f 1 and Der p 1 proteins were isolated a number of years (see, for example, U.S. Pat. No. 5,433,948, issued Jul. 18, 1995, by Thomas et al; U.S. Pat. No. 5,552,142, issued Sep. 3, 1996, by Thomas et al; U.S. Pat. No 5,770,202, issued Jun. 23, 1998, by Thomas et al; U.S. Pat. No.5,773,002, issued Jun. 30, 1998, by Thomas et al; PCT Patent Publication No. WO 88/10297, published Dec. 29, 1988, by Thomas et al; PCT Patent Publication No. WO 92/04445, published Mar. 19, 1992, by Thomas et al); and researchers have tried to express Group 1 proteins in recombinant expression systems. Until the present invention, however, there has been only limited success in producing an active, easily purified recombinant mite Group 1 protein. For example, although researchers have tried to produce active, fully functional recombinant Der f 1 and Der p 1 proteins in E. coli, insect cells, and Saccharomyces cerevisiae, to date, none of these efforts has yielded an easily purified Group 1 protein that binds to IgE in mite-allergic patients in a manner equivalent to a native Group 1 protein.
Using E. coli, researchers expressed a recombinant Der p 1 protein as a fusion protein with glutathione S transferase. The resulting recombinant protein was produced in very low yields, at about 200 micrograms soluble protein per liter of culture medium. Furthermore the GST-Der p 1 fusion protein exhibited only about 50% of the IgE reactivity of the native protein; see Chua et al, 1992, J Allergy Clin Immunol 89, 95-102. Moreover, this protein was expressed using a cDNA encoding the mature form, not the pro-form. The inventors are not aware of any reports of successful expression in E. coli of an active Der p 1 protein encoded by the pro-form.
Using a baculovirus expression system in insect cells, Shoji et al, 1996, Biosci Biotech Biochem 60, 621-625, and Shoji et al, 1997, Biosci Biotech Biochem 61, 1668-1673, reported production of a recombinant Der f 1 protein with IgE binding activity comparable to the native protein, but their process required an additional post-purification step of either acid or enzymatic treatment to cleave the pro-form to a mature form. This cleavage step was necessary because the Der f 1 pro-form had only 20% of the IgE reactivity of the native protein. To avoid the step of acid proteolysis, Shoji et al. converted a glutamate at the carboxyl-terminus of the “pro” region of the pro-form to a lysine residue. This genetically engineered version of Der f 1 protein could then be cleaved with lysylendopeptidase to the mature form under conditions that did not result in the cleavage of internal lysines. However, this process is disadvantageous, especially at large scale, for producing recombinant proteins. Controlling a relatively non-specific protease cleavage step to cleave at only one specific site out of several possible sites is inherently difficult. In addition, use of a protease adds steps and cost to the purification process.
Using the yeast S. cerevisiae, Chua et al, ibid., produced recombinant Der p 1 protein. Although the Der p 1 protein was expressed as the pro-form in this system, the protein was not secreted by S. cerevisiae. As such, the resulting insoluble protein was purified using several steps, including solubilization, renaturation and affinity chromatography; such a process resulted in low yields of only about 1 milligram protein per liter of yeast culture. Furthermore, the recovered S. cerevisiae-expressed Der p 1 proenzyme exhibited only about 80% of the activity of native Der p 1 protein: Of 11 sera tested that were reactive with the native protein, only 9 were reactive with the S. cerevisiae expressed Der p 1 protein.
Thus, there remains a need in the art for an expression system that produces recombinant mite Group 1 proteins that exhibit activity equivalent to that of native mite Group 1 proteins. Preferably such a system would allow for easy and cost-effective recovery of such recombinant proteins.